Engineering a Chromoprotein Optimized for Photoacoustic Imaging and Biosensing Applications

Abstract

A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which serve as important tools in biomedical research. Recently, chromoproteins have been utilized as both reporter molecules in photoacoustic imaging and acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Photoacoustic imaging enables imaging deep in tissue with relatively high resolution while FRET-based biosensors are the principal technology for live cell imaging of physiological events, such as enzymatic activity, protein-protein interaction and changes in small molecule concentration. However, there are few chromoproteins that have ideal characteristics for photoacoustic imaging and biosensors due to a limited ability to artificially evolve them for improved photoacoustic signals. A major challenge of directed laboratory protein evolution is establishing a simple and efficient screening method. In this thesis we describe our efforts to address this shortcoming in the area of chromoproteins evolution and application by developing a novel colony-based photoacoustic screening method. Through iterative rounds of directed evolution and subsequent screening, the best variants of chromoproteins exhibited higher photoacoustic signal and extinction coefficient and lower quantum yield. We also report the application and performance of a tandem dimer chromoprotein in FRET-based biosensors compared with monomer acceptor. The change of donor fluorescence represented the functionality of biosensor attributing to non-fluorescence of acceptor. Specifically, we demonstrated that tandem dimer-based FRET biosensors are useful for detecting activation of caspase-3 and changes in calcium ion (Ca2+) concentration in live cells.